【作者】 李萌；

【导师】 佟倜；

【作者基本信息】 吉林大学 ， 外科学（专业学位）， 2025， 博士

【摘要】 研究背景:肺癌是全球癌症相关死亡的首要原因之一,约占癌症总死亡率的18%。由于早期诊断困难且易发生转移,肺癌已成为临床治疗失败和患者死亡率居高不下的主要原因。深入研究肺癌发生发展的关键生物标志物及其分子机制,不仅有助于阐明肿瘤进展的生物学本质,更为非小细胞肺癌(Non-small cell lung cancer,NSCLC)的个体化精准治疗提供了新的理论依据和创新性治疗策略。近年来,细胞信号通路的研究取得了显著进展,大量关键蛋白分子已被成功鉴定。然而,要更全面地阐明肺癌的分子机制,仍需要提高对亚细胞层面生物过程的认识。细胞膜作为细胞生存和功能执行的关键结构单元,不仅是多种生物化学反应的重要场所,其膜蛋白更在细胞生理过程中起着重要作用。神经纤毛蛋白1(Neuropilin 1,NRP1)是一种多功能的膜受体蛋白。最初在神经元中被发现,后续研究揭示了它在内皮细胞和免疫细胞表面均有表达,参与调控多种生理和病理过程,并在肺发育过程中发挥重要作用,调控肺部分支发育。作为多种配体的共同受体,NRP1的高表达与肿瘤的起始、发展、侵袭、转移和患者的预后紧密相关,已经成为肿瘤靶向治疗的重要靶点。因此,深入研究肺癌细胞膜表面蛋白NRP1的表达差异,对于揭示肿瘤发生发展以及开发实体肿瘤的新型诊疗策略具有重要意义。然而,膜蛋白的分子尺寸通常在数十纳米量级,这一尺度远低于传统光学显微镜的分辨极限。近年来,以直接随机光学重构显微镜(direct Stochastic Optical Reconstruction Microscopy,dSTORM)为代表的超分辨率荧光显微镜技术的突破性进展,成功突破了光学衍射极限,实现了纳米级尺度的超分辨率成像,为膜蛋白的结构与功能研究提供了强有力的技术支撑。本研究首次采用dSTORM成像技术,系统解析了NRP1在NSCLC细胞膜上的空间分布特征。通过定量分析NRP1蛋白的异质性分布模式,我们旨在阐明其在细胞膜上的空间分布规律及其与NSCLC生物学特性的内在关联。同时,本研究将进一步揭示调控NRP1膜分布的功能机制,以阐明其在肺癌发生发展中的作用。该研究不仅有望为NSCLC的分子病理机制提供新的理论依据,也可能为开发靶向治疗策略提供潜在的新型分子靶点。研究方法:在本研究中,我们主要采用dSTORM成像技术,实现了对NSCLC细胞膜表面NRP1蛋白空间分布特征的纳米级解析。为准确处理和分析所获得的图像数据,我们运用Image J和SR-Tesseler软件进行图像重构和定量分析,确保实验数据的可靠性和可重复性。此外,我们还整合了多种传统实验方法,包括生物信息学分析、免疫印迹、CCK-8检测、Ed U染色、平板克隆、细胞划痕以及Transwell等实验。通过这些综合的实验方法,我们旨在深入阐明NRP1在NSCLC中的表达特征、生物学功能及其下游信号转导途径,为揭示NRP1在肺癌发生发展中的分子机制提供坚实的理论依据。在第一部分中,我们首先通过整合多个生物信息学数据库,系统分析了NRP1在NSCLC中蛋白和m RNA的表达谱及其与临床预后的相关性。随后采用dSTORM成像技术,比较了肺癌细胞与正常肺上皮细胞中NRP1蛋白的空间定位特征,并且进一步观察了原代细胞和病理组织的空间分布模式。在第二部分中,我们运用dSTORM成像技术分析了调控NRP1蛋白成簇的多个调控因素。首先,通过分析细胞顶-基底膜极性对NRP1分布的影响,揭示其空间定位特征。其次,探究NRP1与脂筏微环境的关联。随后,通过细胞松弛素B和D处理破坏肌动蛋白细胞骨架,评估其对NRP1分布的影响。此外,为阐明NRP1自身活性对其分布的影响,我们构建了NRP1敲低和过表达细胞模型,系统分析其空间分布特征的变化。同时,解析了NSCLC中NRP1的激活模式,我们重点研究了肝细胞生长因子与NRP1的相互作用,并探讨了其激活状态与细胞内分布特征的潜在关联。最后,我们深入研究了c-MET对NRP1蛋白分布的调控作用。在第三部分中,我们采用了慢病毒介导的基因沉默技术,成功构建了NRP1在A549细胞中的沉默表达模型以及在H1299细胞中的质粒介导的过表达模型。采用CCK-8增殖实验、Ed U染色实验、平板克隆实验、划痕实验以及Transwell迁移和侵袭实验,系统评估了NRP1蛋白簇的改变对NSCLC细胞增殖、迁移和侵袭能力的影响。进一步利用免疫印迹分析了沉默和过表达NRP1的蛋白簇对上皮-间充质转化(Epithelial-Mesenchymal Transition,EMT)相关蛋白及PI3K/AKT/m TOR信号通路下游关键分子的调控机制。研究结果:1、通过整合多个生物信息学数据库,我们发现NRP1在肺癌组织和细胞中的表达水平显著上调,且其高表达与患者的不良预后呈显著正相关。为深入解析NRP1的分布特征,我们采用超分辨率荧光显微技术,系统观察了NRP1在培养细胞系、原代细胞及病理组织切片中的空间分布模式。研究发现,与正常肺细胞相比,肺癌细胞中NRP1蛋白倾向于形成更多且尺寸更大的簇状结构,且其分布模式具有细胞系特异性。这些簇状结构由多个NRP1分子聚集而成,在原代细胞和组织切片中表现得尤为显著,提示NRP1的聚集特性可能与肿瘤的恶性表型密切相关。值得注意的是,组织切片中NRP1蛋白簇的分布呈现明显的空间异质性,这可能反映了肿瘤微环境中复杂的细胞间相互作用和信号转导网络。2、使用dSTORM成像观察到NRP1在顶端膜和基底外侧膜上均呈现簇状分布,但顶端膜上的蛋白簇在数量、体积和密度方面均显著高于基底外侧膜。当脂筏结构被破坏后,NRP1蛋白簇的尺寸和数量均显著减少。双色dSTORM共定位分析进一步证实了NRP1与脂筏存在空间共定位关系。同时,当细胞中的肌动蛋白骨架被破坏后,NRP1蛋白在细胞膜上的分布会减少,其成簇能力受到明显抑制。为探究NRP1活性对其空间分布的调控作用,我们通过基因敲低和过表达技术来调控NRP1的表达水平,发现NRP1表达下调导致细胞膜上蛋白簇的聚集能力减弱,表现为分布分散且尺寸缩小,而NRP1过表达则显著增强了其在细胞膜上的聚集能力。值得注意的是,作为多功能受体,NRP1在肝细胞生长因子刺激下虽然整体聚集趋势有所下降,但更倾向于形成大尺寸蛋白簇。最后,通过双色dSTORM共定位成像和c-MET沉默实验,我们发现NRP1与c-MET存在显著的空间共定位,且c-MET表达下调明显减弱了NRP1的聚集能力,提示c-MET是调控NRP1成簇的关键因子。3、NRP1蛋白在细胞膜上的空间分布模式对肿瘤细胞的恶性表型具有显著调控作用。当NRP1蛋白成簇结构被破坏时,肺癌细胞的增殖能力、克隆形成能力以及迁移侵袭活性均受到显著抑制。同时,EMT相关标志蛋白N-cadherin和Vimentin的表达下调,而上皮标志蛋白E-cadherin的表达上调,且下游信号通路关键蛋白(如AKT、GSK3β和m TOR)的磷酸化水平降低。相反,当NRP1蛋白形成稳定的聚集体时,能够显著促进肺癌细胞的增殖、增强其克隆形成能力并提高迁移侵袭活性。此外,EMT相关蛋白N-cadherin和Vimentin的表达上调,E-cadherin的表达下调,同时下游信号蛋白(如AKT、GSK3β和m TOR)的磷酸化水平显著升高。这些结果表明,NRP1蛋白的成簇状态通过调控EMT进程和下游信号通路活性,在肺癌细胞的恶性表型中发挥关键作用。研究结论:1、NRP1在NSCLC中呈现高表达。研究首次采用dSTORM技术,在单分子水平上发现NRP1在肺癌细胞和组织中呈现显著的蛋白簇状分布,而在正常肺细胞和组织中则表现为散在分布。2、利用dSTORM发现细胞膜表面NRP1蛋白簇的分布特征与细胞极性、脂筏结构、细胞骨架、蛋白表达水平、激活模式及其与c-MET的相互作用密切相关。3、NRP1蛋白簇分布的改变可显著影响肺癌细胞的增殖、迁移和侵袭能力,并通过调控下游信号通路参与NSCLC的疾病进展。更多还原

【Abstract】 Background:Lung cancer stands as one of the leading causes of cancer-related deaths worldwide,accounting for approximately 18%of total cancer mortality.Due to the challenges in early diagnosis and its propensity for metastasis,lung cancer has become a primary reason for clinical treatment failure and persistently high patient mortality rates.In-depth research into the key biomarkers and molecular mechanisms underlying the development and progression of lung cancer not only helps to elucidate the biological essence of tumor progression but also provides new theoretical foundations and innovative therapeutic strategies for the personalized precision treatment of non-small cell lung cancer(NSCLC).In recent years,significant progress has been made in the study of cellular signaling pathways,and a large number of key protein molecules have been successfully identified.However,to more comprehensively elucidate the molecular mechanisms of lung cancer,it is still necessary to enhance our understanding of biological processes at the subcellular level.As a critical structural unit for cell survival and function,the cell membrane is not only an important site for various biochemical reactions,but its membrane proteins also play a crucial role in cellular physiological processes.Neuropilin 1(NRP1)is a multifunctional membrane receptor protein.Initially identified in neurons,subsequent studies have revealed its expression on the surface of endothelial cells and immune cells,where it participates in regulating various physiological and pathological processes.NRP1 plays a crucial role in lung development,particularly in regulating lung branching morphogenesis.As a co-receptor for multiple ligands,the high expression of NRP1 is closely associated with tumor initiation,progression,invasion,metastasis,and patient prognosis.Due to its significant role in tumor biology,NRP1 has emerged as an important target for tumor-targeted therapy.Therefore,in-depth research into the differential expression of the membrane protein NRP1 in lung cancer cells is of great significance for elucidating tumorigenesis and progression,as well as for developing novel diagnostic and therapeutic strategies for solid tumors.However,the molecular size of membrane proteins typically ranges in the tens of nanometers,a scale far below the resolution limit of conventional optical microscopy.In recent years,breakthrough advancements in super-resolution fluorescence microscopy technologies,represented by direct Stochastic Optical Reconstruction Microscopy(dSTORM),have successfully surpassed the optical diffraction limit,enabling super-resolution imaging at the nanoscale.This provides powerful technical support for the study of the structure and function of membrane proteins.This study is the first to employ dSTORM imaging technology to systematically analyze the spatial distribution characteristics of NRP1 on the cell membrane of NSCLC cells.Through quantitative analysis of the heterogeneous distribution patterns of NRP1 proteins,we aim to elucidate their spatial distribution patterns on the cell membrane and their intrinsic relationship with the biological properties of NSCLC.Additionally,this research seeks to uncover the functional mechanisms regulating the membrane distribution of NRP1,thereby clarifying its role in the development and progression of lung cancer.This research not only holds the potential to provide new theoretical insights into the molecular pathological mechanisms of NSCLC but may also offer novel molecular targets for the development of targeted therapeutic strategies.Research Methods:In this study,we primarily utilized dSTORM imaging technology to achieve nanoscale resolution of the spatial distribution characteristics of NRP1 protein on the membrane of NSCLC cells.To accurately process and analyze the obtained image data,we employed Image J and SR-Tesseler software for image reconstruction and quantitative analysis,ensuring the reliability and reproducibility of the experimental data.Additionally,we integrated various traditional experimental methods,including bioinformatics analysis,Western blotting,CCK-8 assay,Ed U staining,plate cloning,cell scratch assay,and Transwell experiments.Through these comprehensive experimental approaches,we aim to elucidate the expression characteristics,biological functions,and downstream signaling pathways of NRP1 in NSCLC,providing a solid theoretical foundation for understanding the molecular mechanisms of NRP1 in the development and progression of lung cancer.In the first part,we initially integrated multiple bioinformatics databases to systematically analyze the protein and m RNA expression profiles of NRP1 in NSCLC and their correlation with clinical prognosis.Subsequently,we employed dSTORM imaging technology to compare the spatial localization characteristics of NRP1 protein between lung cancer cells and normal lung epithelial cells.Furthermore,we observed the spatial distribution patterns in primary cells and pathological tissues.In the second part,we utilized dSTORM imaging technology to analyze multiple regulatory factors influencing the clustering of NRP1 protein.Firstly,by examining the impact of apical-basal polarity on NRP1 distribution,we revealed its spatial localization characteristics.Secondly,we investigated the association between NRP1 and the lipid raft microenvironment.Subsequently,we disrupted the actin cytoskeleton using cytochalasin B and D treatments to assess their effects on NRP1 distribution.Additionally,to elucidate the influence of NRP1’s own activity on its distribution,we constructed NRP1 knockdown and overexpression cell models,systematically analyzing changes in its spatial distribution characteristics.Furthermore,to decipher the activation patterns of NRP1 in NSCLC,we focused on the interaction between hepatocyte growth factor and NRP1,exploring the potential correlation between its activation state and intracellular distribution features.Finally,we delved into the regulatory role of c-MET on NRP1 protein distribution.In the third part,we employed lentivirus-mediated gene silencing technology to successfully construct a knockdown model of NRP1 in A549 cells and a plasmid-mediated overexpression model in H1299 cells.Using CCK-8 proliferation assays,Ed U staining assays,plate cloning assays,scratch assays,and Transwell migration and invasion assays,we systematically evaluated the impact of altered NRP1 protein clustering on the proliferation,migration,and invasion capabilities of NSCLC cells.Furthermore,Western blot analysis was utilized to investigate the regulatory mechanisms of NRP1 protein clusters,both silenced and overexpressed,on epithelial-mesenchymal transition(EMT)-related proteins and key downstream molecules of the PI3K/AKT/m TOR signaling pathway.Results:1.By integrating multiple bioinformatics databases,we found that the expression level of NRP1 is significantly upregulated in lung cancer tissues and cells,and its high expression is positively correlated with poor patient prognosis.To further analyze the distribution characteristics of NRP1,we employed super-resolution fluorescence microscopy to systematically observe the spatial distribution patterns of NRP1 in cultured cell lines,primary cells,and pathological tissue sections.The study revealed that,compared to normal lung cells,NRP1 proteins in lung cancer cells tend to form more and larger clusters,with distribution patterns being cell line-specific.These clusters are composed of multiple NRP1 molecules and are particularly prominent in primary cells and tissue sections,suggesting that the clustering behavior of NRP1 may be closely related to the malignant phenotype of tumors.Notably,the distribution of NRP1 protein clusters in tissue sections exhibits significant spatial heterogeneity,which may reflect the complex intercellular interactions and signaling networks within the tumor microenvironment.2.Using dSTORM imaging,we observed that NRP1 exhibits clustered distribution on both the apical and basolateral membranes,but the number,size,and density of protein clusters on the apical membrane were significantly higher than those on the basolateral membrane.When the lipid raft structure was disrupted,both the size and number of NRP1 protein clusters were significantly reduced.Dual-color dSTORM co-localization analysis further confirmed the spatial co-localization relationship between NRP1 and lipid rafts.Additionally,when the actin cytoskeleton in cells was disrupted,the distribution of NRP1 on the cell membrane decreased,and its clustering ability was significantly inhibited.To investigate the regulatory role of NRP1 activity on its spatial distribution,we modulated NRP1 expression levels through gene knockdown and overexpression techniques.We found that downregulation of NRP1 expression led to a reduction in the clustering ability of protein clusters on the cell membrane,manifesting as a dispersed distribution and smaller cluster sizes.Conversely,NRP1 overexpression significantly enhanced its clustering ability on the cell membrane.Notably,as a multifunctional receptor,NRP1exhibited an overall decrease in clustering tendency under hepatocyte growth factor stimulation but showed a preference for forming larger protein clusters.Finally,through dual-color dSTORM co-localization imaging and c-MET knockdown experiments,we observed significant spatial co-localization between NRP1 and c-MET.Downregulation of c-MET expression markedly weakened the clustering ability of NRP1,suggesting that c-MET is a key factor regulating NRP1 clustering.3.The spatial distribution pattern of NRP1 protein on the cell membrane significantly regulates the malignant phenotype of tumor cells.When the clustering structure of NRP1 protein is disrupted,the proliferation capacity,clonogenic ability,and migratory and invasive activities of lung cancer cells are markedly inhibited.Concurrently,the expression of EMT-related marker proteins,N-cadherin and Vimentin,is downregulated,while the expression of the epithelial marker protein E-cadherin is upregulated.Additionally,the phosphorylation levels of key downstream signaling pathway proteins(such as AKT,GSK3β,and m TOR)are reduced.Conversely,when NRP1 protein forms stable aggregates,it significantly promotes the proliferation,enhances the clonogenic ability,and increases the migratory and invasive activities of lung cancer cells.Furthermore,the expression of EMT-related proteins N-cadherin and Vimentin is upregulated,while the expression of E-cadherin is downregulated,and the phosphorylation levels of downstream signaling proteins(such as AKT,GSK3β,and m TOR)are significantly elevated.These results indicate that the clustering state of NRP1 protein plays a crucial role in the malignant phenotype of lung cancer cells by regulating the EMT process and the activity of downstream signaling pathways.Conclusions:1.NRP1 is highly expressed in NSCLC.For the first time,dSTORM technology was used to reveal that NRP1 exhibits significant protein clustering in lung cancer cells and tissues at the single-molecular level,whereas it shows scattered distribution in normal lung cells and tissues.2.Using dSTORM,it was discovered that the distribution characteristics of NRP1 protein clusters on the cell membrane are closely related to cell polarity,lipid raft structure,cytoskeleton,protein expression levels,activation status,and its interaction with c-MET.3.Alterations in the distribution of NRP1 protein clusters can significantly affect the proliferation,migration,and invasion capabilities of lung cancer cells and participate in the progression of NSCLC by regulating downstream signaling pathways.更多还原